Bone remodeling is a sequence of events in which osteoclastic resorption is coupled to osteoblast-driven formation. Resorption releases Ca2+, matrix proteins, growth factors, and signaling molecules into the microenvironment. Cells in bone and marrow respond to fluxes in the [Ca2+]e which initiate signaling cascades. In osteoblasts, changes in the [Ca2+]e affect expression of genes critical for matrix production and mineralization, chemotaxis, and proliferation. Considerable evidence supports the idea that changes in the [Ca2+]e couple to the activation of CaRs. CaRs are known to control PTH secretion, parathyroid cell growth, and renal Ca2+ and water handling. These aspects of CaR physiology were confirmed in the original (global) CaR knockout mouse developed by Ho et al in 1995. This knockout was generated by a targeting strategy that was later learned to allow for the production of alternatively spliced CaRs that are in fact expressed in bone, marrow, cartilage, skin and other cells. Thus, to understand the role of CaRs in bone cells and skeletal homeostasis in vivo, a different knockout model is needed -- one that selectively targets bone cell populations (conditional knockout) and that does not allow for the formation of any membrane-anchored, signaling-competent CaRs. Our laboratory recently developed a novel floxed CaR mouse that meets these specifications. We propose to use this model and osteoblast- specific Tg mice expressing Cre recombinase under the control of specific bone promoters (type 1 collagen, osteocalcin, osterix, dentin matrix protein 1) to develop conditional knockouts of the CaR targeted to bone cell populations at different stages of differentiation. We will test the hypothesis that CaRs mediate high [Ca2+]e-induced signaling and changes in differentiation, gene expression, and mineralization in osteoblasts, that CaRs act at specific points temporally in bone development, and that CaR activation mediates its effects at least partly via the Wnt/-catenin pathway. Our aims are (1) to determine the role of the CaR in the growth and differentiation of cells in the osteoblastic lineage by analyzing mice with conditional deletion of the CaR at temporally different points in osteoblastic development; and (2) to determine whether cellular and phenotypic features of knocking-out the CaR in osteoblasts are mediated via Wnt/-catenin signaling. Skeletal phenotypes in vivo will be characterized by in vivo micro-CT, histomorphometry, biochemical markers of turnover, and serum chemistry and hormonal parameters. Cells from these animals will be cultured to determine the key genes and signaling pathways affected. These studies should lay the groundwork for developing therapeutics targeted to CaRs in different bone cell populations as a means to treat disorders of low bone formation and excessive remodeling such as osteoporosis, hyperparathyroidism, and bone metastases. PUBLIC HEALTH RELEVANCE: Calcium-sensing receptors are molecules on the membranes of cells that sense the level of calcium in the environment and communicate this information to the inside of the cell. Fluctuations in the concentration of calcium act as a signal to bone cells that cause them to change their responses and their activities. The goal of this proposal is to figure out what calcium is doing in bone cells by removing calcium-sensing receptors selectively from the different types of cells in bone in a mouse model, to better understand bone metabolism and bone diseases, and to find more effective treatments.